The discovery over the past decade of cellular ocogenes has provided one explanation of the molecular mechanisms that are responsible for the neoplastic conversion of many types of cells.
Nevertheless, these genes and similarly acting cellular elements can at best explain only part of the process of tumor formation. Before growing out into a tumor, the transformed cells must confront and evade physiological mechanisms that are designed to defend the host against cancer.
Prominent in these defenses, presumably are immune mechanisms that involve specific recognition and elimination of tumor cells. These mechanisms are poorly understood: the nature and importance of immunological effector mechanisms are unclear, and the identity of tumor cell markers that may be recognized by these effectors remains mostly elusive.
A desirable mode of cancer treatment is to enlist natural immune mechanisms to establish anti-tumor immunity. Methods for inducing effective anti-tumor immunity, however, remain to be elucidated. One possible way of inducing immune response against a tumor might be to immunize with a tumor-associated antigen. For examples, the ectodomain of the neu-encoded rat p185 protein constitutes a highly immunogenic determinant in tumor-bearing NFS mice which invariably mount a strong serum response to this protein. See, Padhy, L. C. et al. (1982) Cell, 28:865-871. Tumors formed from neu-trans-fectants (cells transformed with the neu gene) initially grow rapidly but ultimately are seen to regress. This regression is not seen with tumors formed from other types of oncogene-transfected cells and can be attributed at least partially to recognition of the neu-transfectants by the host immune system. The nature of the immune mechanisms that effect this regression and establish anti-tumor immunity is unclear. It is possible that the p185 antigen alone suffices to induce the anti-tumor response. Alternatively, this antigen may only provoke an effective response when acting in concert with other unrelated, transformation-specific antigens displayed by the oncogene-transformed cells.
A purified, murine melanoma tumor-specific antigen has been demonstrated to elicit tumor rejection of a melanoma. Hearing, V. J. et al. J. Immunol., 137:379 (1986). This work suggests that tumor-associated antigens can be used successfully as targets for tumor immunotherapy. Recently, Lathe et al. showed that immunization of mice with a recombinant vaccinia virus capable of expressing a polyoma-virus encoded antigen induced rejection of viral-induced tumor. Lathe et al. (1987) Nature 326, 878-880. The polyoma viral antigen, however, is a completely foreign and highly immunogenic antigen.
This invention pertains to recombinant pox viruses capable of expressing cell-encoded tumor-associated antigens, to methods of producing the recombinant pox virus, to intermediate DNA vectors which recombine with pox virus in vivo to produce the modified pox viruses and to methods of immunizing a host with the recombinant pox virus to elicit an immune response against a cell-encoded tumor-associated antigen. The invention is based, in part, on the discovery that immunization with the neu antigen via a recombinant pox virus serves as effective prophylaxis against tumors formed by neu oncogene-transfected cells.
Recombinant pox virus capable of expressing a cell-encoded tumor-associated antigen are produced by integrating into the pox virus genome sequences encoding the antigen or immunogenic portions thereof. Tumor-associated antigens can be cellular oncogene-encoded products or aberrantly expressed proto-oncogene-encoded products (e.g. products encoded by the neu, ros, trk, and kit genes) and mutated forms of growth factor receptor or receptor-like cell surface molecules (e.g. surface receptor encoded by the c-erb B gene). Other tumor-associated antigens include molecules which may or may not be directly involved in transformation events, but are expressed by tumor cells (e.g. carcinoembryonic antigen, CA-125, melonoma associated antigens, etc.) In some embodiments the sequence encoding the tumor-associated antigen is engineered to encode a product which retains at least an immunogenic domain but is disabled with respect to its oncogenic activity. For example, truncated products may be designed which contain the immunogenic domains of the natural gene product but which either lack or contain inactivated oncogenic regions.
The DNA sequence encoding the tumor-associated antigen is inserted into a region of the pox virus genome which is nonessential for replication of the pox virus, generally in association with a pox virus promoter to direct its expression.
The DNA-sequence encoding the tumor-associated antigen is integrated into the pox viral genome by an in vivo recombination event between an intermediate DNA vector carrying the DNA encoding the tumor-associated antigen and a pox virus. In essence, the intermediate DNA vector contains the antigen-encoding sequence linked to a pox viral promoter located within a DNA sequence homologous to a region of the pox viral genome which is nonessential for replication of the pox virus. Thus, at minimum the vector comprises:
a. a prokaryotic origin of replication;
b. a pox viral promoter;
c. a sequence encoding a tumor-associated antigen under the direction of the pox viral promoter; and
d. DNA sequences of the pox virus into which the gene encoding the antigen sequence is to be integrated, the DNA sequences flanking the promoter and structural gene at both the 5xe2x80x2 and 3xe2x80x2 end, the DNA sequence being homologous to the region of the pox virus genome where the sequence of the tumor associated antigen is to be inserted.
Recombination of the DNA vector and the pox virus is achieved in an appropriate host cell. Appropriate host cells for in vivo recombination are eukaryotic cells which are 1) transfectable by the DNA vector and 2) infectable by pox virus. The host cell is transfected with the DNA vector carrying the antigen sequence and then infected with the pox virus. The virus is allowed to replicate in the host cell during which time recombination occurs in vivo between the DNA vector and the virus resulting in insertion of the sequence encoding the tumor-associated antigen into the pox virus genome. The recombinant viral progeny is isolated from the wild type virus.
An assayable marker can be co-integrated with the antigen-encoding sequence. Expression of the marker provides a basis for selection of recombinant virus containing integrated DNA. Other methods of selection include detection of the integrated sequences by hybridization with homologous DNA probes. Negative selection procedures can also be used such as selection for absence of the product of the viral gene into which the DNA segment has been inserted. When an assayable marker is located at the viral insertion site, recombinants can be identified by loss of the marker.
The recombinant virus is a virus which expresses in an inoculated host the cellular tumor-associated antigen. The virally-expressed product will trigger cell-mediated and/or humoral immunity against the antigen and cells bearing the antigen.